Nutrient-regulated dynamics of chondroprogenitors in the postnatal murine growth plate

Longitudinal bone growth relies on endochondral ossification in the cartilaginous growth plate, where chondrocytes accumulate and synthesize the matrix scaffold that is replaced by bone. The chondroprogenitors in the resting zone maintain the continuous turnover of chondrocytes in the growth plate. Malnutrition is a leading cause of growth retardation in children; however, after recovery from nutrient deprivation, bone growth is accelerated beyond the normal rate, a phenomenon termed catch-up growth. Although nutritional status is a known regulator of long bone growth, it is largely unknown whether and how chondroprogenitor cells respond to deviations in nutrient availability. Here, using fate-mapping analysis in Axin2CreERT2 mice, we showed that dietary restriction increased the number of Axin2+ chondroprogenitors in the resting zone and simultaneously inhibited their differentiation. Once nutrient deficiency was resolved, the accumulated chondroprogenitor cells immediately restarted differentiation and formed chondrocyte columns, contributing to accelerated growth. Furthermore, we showed that nutrient deprivation reduced the level of phosphorylated Akt in the resting zone and that exogenous IGF-1 restored the phosphorylated Akt level and stimulated differentiation of the pooled chondroprogenitors, decreasing their numbers. Our study of Axin2CreERT2 revealed that nutrient availability regulates the balance between accumulation and differentiation of chondroprogenitors in the growth plate and further demonstrated that IGF-1 partially mediates this regulation by promoting the committed differentiation of chondroprogenitor cells.

μm in depth, were cropped out and analyzed in surpass and slice modes of Imaris. Chondrocyte columns were defined as aggregations comprising of four or more cells. Columns comprising nine or fewer cells were denoted as a short column, and those containing 10 or more cells as long columns. 3 The number of chondrocyte columns was counted manually in 3D using Imaris software. Three serial 2D images with a 10 μm interval per sample were used to quantify the number of ZsGreen + cells in the top 50μm 4 and the percentage of ZsGreen + cells among the growth plate chondrocytes. The length of the resting zone changes with age, and has been reported in ranges 40-60 μm at 2-6 weeks of ages. 5 To avoid complexity of defining the resting zone in individual sections, we counted the number of ZsGreen + cells in the top 50 μm to quantify the ZsGreen + cells in the resting zone. For the analysis of ZsGreen + cells in the resting zone at P3, the ZsGreen + cell rate was calculated in a square area measuring 200 µm per side, in a region where round chondrocytes were observed (Fig.1a). Quantification was performed using ImageJ software. Detection and imaging for H&E staining, alizarin labeling, and EdU labeling were performed using Keyence BZX710 (Keyence) and quantification was performed using a BZ-X Analyzer (Keyence).
Histological analysis. Histological analyses were performed using the results of H&E staining, which was performed according to standard protocols. The central area of the proximal tibial growth plate with a width of 600 µm was cropped and analyzed. Growth plate height was measured parallel to the chondrocyte column.
The resting and proliferative zones were defined by morphology following the method described previously. 5 The resting zone was defined from the lower margin of the secondary ossification center to the slightly flat doublet chondrocytes before formation of long columns of proliferative chondrocytes.
Hypertrophic chondrocytes were defined by a height ≥ 10 μm as reported previously. 5 Growth plate height and hypertrophic zone height were measured at four equally spaced points in the cropped area. Terminal hypertrophic chondrocyte was defined as cells in the last lacuna that were not invaded by metaphyseal blood vessels, 5 and the number of terminal hypertrophic chondrocytes per 100 µm was defined as the column number per 100 µm width.
Assessment of bone growth rate by alizarin injection. To assess the rate of physical bone growth, we injected alizarin subcutaneously (30 μg/g body weight; A3883, Sigma-Aldrich) in mice at the indicated time points. Mice were euthanized 48 h after injection, and undecalcified sagittal knee sections (5 µm in thickness) were prepared. Longitudinal bone growth was evaluated as the distance between the edge of the red fluorescence-labeled metaphysis and the chondro-osseous junction. 5 The distances at four equally spaced points in a center area of the proximal tibial growth plate with a width of 600 µm were measured per section.
The sections were examined under 40x magnification for p-H3 and 60x magnification for p-Akt. A central area of the proximal tibial growth plate with a width of 600 µm was selected, and the ratio of p-H3 + cells or p-Akt + cells among DAPI + or ZsGreen + cells was analyzed using Image J. For Igf-1 immunostaining, decalcified sagittal knee paraffin sections (6 µm in thickness) were prepared. Antigen retrieval was performed by incubating the sections with 10 mM sodium citrate (pH 6.0) for 10 min at 80C°. After blocking with 5% goat serum (G9023, Sigma-Aldrich) in PBS for 30 min, sections were incubated with rabbit anti-IGF1 antibody (1:100; ab9572, Abcam) for overnight at 4C°, washed with PBS, and subsequently incubated with Alexa Fluor 649-conjugated goat anti-rabbit IgG (1:200; A11011, Thermo Fisher) for 60 min at room temperature.
In situ hybridization. RNA in situ hybridization was performed on proximal tibial growth plates as described previously. 6 Briefly, undecalcified sagittal knee sections (5 µm in thickness) were post-fixed in freshly prepared 2% formaldehyde for 30 min at room temperature, dehydrated with 70% ethanol for at least one day at 4 °C. The sections on the film were then placed on glass slides. Once the ethanol volatilized, the four sides of the film were surrounded with vacuum grease dissolved in spectroscopic-grade chloroform (C298-500, Sigma-Aldrich) to fix the sections onto glass slides. After the grease was dried, RNA in situ hybridization was performed using RNAscope 2. growth plate with a width of 600 µm was used for analysis. We performed quantification according to the semi-quantitative scoring guideline from the manufacturer: Score 0, no staining or <1 dot/10 cells; Score 1, 1-3 dots/cell; Score 2, 4-9 dots/cell, no or very few dot clusters; Score 3, 10-15 dots/cell and/or <10% of dots in clusters; Score 4, > 15 dots/cell and/or >10% of dots in clusters (https://acdbio.com/dataanalysisguide). For genes with low expression levels, such as Ki67, Cd73, and Pthrp, the sections were examined under 40x magnification and the cells with at least one dot (Score 1-4) were counted as positive. For genes with relatively high expression levels, such as Igf-1 and Clu, the sections were examined under a 20x magnification and the cells with more than 10 dots or with clustering dots (Score 3, 4) were counted as positive.
EdU labelling and detection. To evaluate cell proliferation, EdU (50 μg/g body weight, 900584, Sigma-Aldrich) dissolved in saline was subcutaneously administered to the mice at the indicated postnatal days.
After incubating samples with 0.5% Triton X-100 (9002-93-1, Sigma-Aldrich) in PBS for 10 min at room temperature, the Click-iT EdU Alexa Fluor 647 Imaging Kit (C10340, Thermo Fisher) was used to detect EdU. A central area of the proximal tibial growth plate with a 600 µm width was used for analysis. The ratio of the number of EdU + cells among DAPI + cells was measured by BZ-X Analyzer.
Measurement of serum IGF-1 levels. Serum IGF-1 levels were determined using a mouse-specific Quantikine ELISA kit (R&D Systems, MG100) according to manufacturer protocol.
LMD and RNA-Seq analysis. We performed LMD using snap frozen sections of distal femur and proximal tibial growth plates as previously described. 7 Briefly, an adhesive film was attached to the specimen and 8 µm sections were cut. Frozen specimens were immediately soaked in 80% ethanol for 30 s, then in 100% ethanol for 1 min (twice), then in xylene for 5 min at room temperature, and subsequently dried for 5 min.
Because most Axin2-CreER + cells remained morphologically in the resting zone after seven days of chase ( Fig. 2B), we utilized Axin2Cre ERT2 ;R26R reporter system as a tool to label the resting chondrocytes. We used Axin2Cre ERT2 ;R26R TdTomato mice instead of Axin2Cre ERT2 ;R26R ZsGreen mice because ZsGreen signal was diminished during ethanol fixation. 8 Knee samples were prepared from Axin2Cre ERT2 ;R26R TdTomato mice on (version 36_49) was used to trim/filter low quality sequences using the "qtrim=lr trimq=10 maq=10" option.
Next, the filtered were aligned to the mouse reference genome (GRCm38) using HISAT2 (version 2.1.0) by applying the -no-mixed and -no-discordant options. The reads were summarized for each gene using HTseq-count (version 0.11.2) supplemented with Ensembl gene annotation (GRCm38.78). The edgeR R package was used to fit the read counts to a negative binomial model along with the generalized linear model, and differentially expressed genes were determined by the likelihood ratio test method in edgeR.
Significance was defined to be those with q-value < 0.01 calculated by the Benjamini-Hochberg method to control the false discovery rate (FDR) and log2 fold change is greater than 1 or smaller than -1 in resting chondrocytes compared to proliferative chondrocytes. The ggpubr R package was used to generate a MA plot. The top 500 DEGs were selected, and the ComplexHeatmap R package was used to generate a heatmap.
The list of DEGs was subjected to KEGG pathway analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) bioinformatics resource (david.ncifcrf.gov). We also used the Upstream Regulator Analysis tool in Ingenuity Pathways Analysis Software (version 01-20-04) (Ingenuity Systems Inc., Redwood, CA, USA) to predict the potential cause of the changes in gene expression between resting and proliferative chondrocytes.
Chondrocyte isolation and culture. Chondrocytes were isolated from the growth plate following the protocol previously described with some modifications. 9 Briefly, the femur and tibia bones were incubated in calcium-and magnesium-free Hank's Balanced Salt Solution (HBSS; H6648, Sigma) containing 2 units of liberase TM (Roche) at 37°C for 30 minutes. After removing soft tissues including perichondrium, distal femur, and proximal tibia bones were dislodged from the the diaphyseal end at the boundary between the primary spongiosa and the growth plate. These epiphyseal bones containing the growth plate were then digested in HBSS with 2 units of liberase TM at 37°C for 30 minutes on a rotor. The first digestion solution was collected and centrifuged at 500 g at 4°C for 4 minutes to collect cells followed by additional four

S12
immunostaining of IGF1(magenta) and Axin2+ cells (green) in the proximal tibial growth plate (n=3). The white dashed lines demarcate the growth plate from the secondary ossification center (left). Scale bars: 50 μm.
Supplementary Table S1. Upstream regulator analysis of the genes changing from resting to proliferative chondrocytes in the growth plate cartilage of 30-day-old mice using Ingenuity Pathway Analysis.
Upstream Regulator Activation z-score P value